Method and composition for selectively inhibiting melanoma

ABSTRACT

A composition and method of preventing or inhibiting tumor growth, and of treating malignant melanoma, without toxic side effects are disclosed. Betulinic acid or a betulinic acid derivative is the active compound of the composition, which is topically applied to the situs of tumor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 08/858,011, filed May 16, 1997, now U.S. Pat. No. 5,869,535,which is a continuation-in-part application of U.S. patent applicationSer. No. 08/407,756, filed Mar. 21, 1995, now U.S. Pat. No. 5,658,497filed Aug. 19, 1997.

This invention was made with government support under U01 CA52956awarded by the National Cancer Institute. The government has certainrights in the invention.

FIELD OF THE INVENTION

This invention relates to compositions and methods of selectivelyinhibiting tumors and, more particularly, to treating a malignantmelanoma using plant-derived compounds and derivatives thereof.

BACKGROUND OF THE INVENTION

Over the past four decades the incidence of melanoma has been increasingat a higher rate than any other type of cancer. It is now theorized thatone in 90 American Caucasians will develop malignant melanoma in theirlifetime. While an increasing proportion of melanomas are diagnosedsufficiently early to respond to surgical treatment and achieve agreater than 90% ten-year survival rate, it is estimated that nearly7,000 individuals suffering from metastatic melanoma will die in theUnited States this year.

For patients with metastatic melanoma not amenable to surgicalextirpation, treatment options are limited.5-(3,3-Dimethyl-1-triazenyl)-1-H-imidaz-ole-4-carboxamide (dacarbazine,DTIC) is the most efficacious single chemotherapeutic agent for melanomahaving an overall response rate of 24%. But the duration of response toDTIC is generally quite poor. Combination therapy with other syntheticand recombinant agents, including N,N′-bis(2-chloroethyl)-N-nitrosurea(carmustine, BCNU), cisplatin, tamoxifen, interferon-alpha (INF-α) andinterleukin-2 (IL-2), has a higher response rate (e.g., 30-50%) in sometrials, but a durable complete response rate is uncommon and toxicity isincreased. Sequential chemotherapy has promise, but, clearly, currenttreatment options for individuals suffering from metastatic melanoma areunsatisfactory.

Various drugs derived from natural products, such as adriamycin(doxorubicin) derivatives, bleomycin, etoposide, and vincristine, andtheir derivatives, have been tested for efficacy against melanoma eitheras single agents or in combination therapy. However, similar to thesynthetic and recombinant compounds, these compounds exhibit lowresponse rates, transient complete responses, and high toxicities.

Nonetheless, as demonstrated by known and presently-used cancerchemotherapeutic agents, plant-derived natural products are a provensource of effective drugs. Two such useful natural product drugs arepaclitaxel (taxol) and camptothecin. Paclitaxel originally derived fromthe bark of the Pacific yew tree Taxus brevifolia Nutt. (Taxaceae),currently is used for the treatment of refractory or residual ovariancancer. More recently, clinical trials have been performed toinvestigate the possible role of paclitaxel in the treatment ofmetastatic melanoma. As a single agent, taxol displays activitycomparable to cisplatin and IL-2. Taxol functions by a unique mode ofaction, and promotes the polymerization of tubulin. Thus, the antitumorresponse mediated by taxol is due to its antimitotic activity. Thesecond drug of prominence, camptothecin, was isolated from the stem barkof a Chinese tree, Camptotheca acuminata Decaisne (Nyssaceae).Camptothecin also functions by a novel mechanism of action, i.e., theinhibition of topoisomerase I. Phase II trials of a water-solublecamptothecin pro-drug analog, Irinotican (CPT-11), have been completedin Japan against a variety of tumors with response rates ranging from 0%(lymphoma) to 50% (small cell lung). Topotecan, another water-solublecamptothecin analog, currently is undergoing Phase II clinical trials inthe United States.

Previous antitumor data from various animal models utilizing betulinicacid have been extremely variable and apparently inconsistent. Forexample, betulinic acid was reported to demonstrate dosedependentactivity against the Walker 256 murine carcinosarcoma tumor system atdose levels of 300 and 500 mg/kg (milligrams per kilogram) body weight.In contrast, a subsequent report indicated the compound was inactive inthe Walker 256 (400 mg/kg) and in the L1210 murine lymphocytic leukemia(200 mg/kg) models. Tests conducted at the National Cancer Instituteconfirmed these negative data.

Similarly, antitumor activity of betulinic acid in the P-388 murinelymphocyte test system has been suggested. However, activity was notsupported by tests conducted by the National Cancer Institute. Morerecently, betulinic acid was shown to block phorbol ester-inducedinflammation and epidermal ornithine decarboxylase accumulation in themouse ear model. Consistent with these observations, the carcinogenicresponse in the two-stage mouse skin model was inhibited. Thus, someweak indications of antitumor activity by betulinic acid have beenreported, but, until the present invention, no previous reports or datasuggested that betulinic acid was useful for the selective control ortreatment of human melanoma. Furthermore, to date, no information hasbeen published with respect to the selective activity of derivatives ofbetulinic acid against melanoma cells.

SUMMARY OF THE INVENTION

The present invention is directed to a method and composition forpreventing or inhibiting tumor growth. The active compound is betulinicacid or a derivative of betulinic acid. The betulinic acid is isolatedby a method comprising the steps of preparing an extract from the stembark of Ziziphus mauritiana and isolating the betulinic acid.Alternatively, betulin can be isolated from the extract and used asprecursor for betulinic acid, which is prepared from betulin by a seriesof synthetic steps. The betulinic acid can be isolated from the extractby mediating a selective cytotoxic profile against human melanoma in asubject panel of human cancer cell lines, conducting a bioassay-directedfractionation based on the profile of biological activity using culturedhuman melanoma cells (MEL-2) as the monitor, and obtaining betulinicacid therefrom as the active compound. The resulting betulinic acid canbe used to prevent or inhibit tumor growth, or can be converted to aderivative to prevent or inhibit tumor growth.

An important aspect of the present invention, therefore, is to provide amethod and composition for preventing or inhibiting tumor growth and,particularly, for preventing or inhibiting the growth of melanoma usinga natural product-derived compound, or a derivative thereof.

Another aspect of the present invention is to provide a treatment methodusing betulinic acid to prevent the growth or spread of cancerous cells,wherein the betulinic acid, or a derivative thereof, is applied in atopical preparation.

Another aspect of the present invention is to overcome the problem ofhigh mammalian toxicity associated with synthetic anticancer agents byusing a natural product-derived compound, e.g., betulinic acid or aderivative thereof.

Still another aspect of the present invention is to overcome the problemof insufficient availability associated with synthetic anticancer agentsby utilizing readily available, and naturally occurring betulinic acid,or a derivative thereof.

Yet another aspect of the present invention is to prepare derivatives ofbetulinic acid that have a highly selective activity against melanomacells, and that have physical properties that make the derivativeseasier to incorporate into topical preparations useful for theprevention or inhibition of melanoma cell growth.

These and other aspects of the present invention will become apparentfrom the following description of the invention, which are intended tolimit neither the spirit or scope of the invention but are only offeredas illustrations of the preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of mean tumor volume (in cubic centimeters (cm³)) vs.time for nonestablished MEL-2 tumors in control mice and mice treatedwith increasing dosages of betulinic acid;

FIG. 2 is a plot of mean tumor volume (in cm³) vs. time for establishedMEL-2 tumors in control mice and mice treated with DTIC or betulinicacid;

FIG. 3(A) is a plot of the 50 Kbp (kilobase pairs) band as % total DNAv. time for treatment of MEL-2 cells with 2 μg/ml (micrograms permilliliter) betulinic acid;

FIG. 3(B) is a plot of the 50 Kbp band as % total DNA versusconcentration of betulinic acid (μg/ml); and

FIGS. 4 and 5 are plots of mean tumor volume (cm³) vs. time forestablished and nonestablished MEL-1 tumors in control mice and micetreated with increasing doses of betulinic acid.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Betulinic acid, 3β-hydroxy-lup-20(29)-ene-28-oic acid, is a naturalproduct isolated from several genus of higher plants. Through abioassay-directed fractionation of the stem bark of Ziziphus mauritianaLam. (Rhamnaceae), betulinic acid, a pentacyclic triterpene, wasisolated as an active compound that showed a selective cytotoxicityagainst cultured human melanoma cells. The cell lines evaluated forcytotoxicity were A431 (squamous), BC-1 (breast), COL-2 (colon), HT-1080(sarcoma), KB (human oral epidermoid carcinoma), LNCaP (prostate), LU-1(lung), U373 (glioma), and MEL-1, -2, -3, and -4 (melanoma). Betulinicacid was found to be an excellent antitumor compound against humanmelanoma due to its unique in vitro and in vivo cytotoxicity profile.Betulinic acid has shown a strong selective antitumor activity againstmelanoma by induction of apoptosis. The selective cytotoxicity ofbetulinic acid, and its lack of toxicity toward normal cells, afford afavorable therapeutic index. In addition, betulinic acid has beenreported to have an anti-HIV activity.

The bark of white birch, Betula alba, contains betulin (up to about25%), lup-20(29)-ene-3β,28-diol, and betulinic acid (0.025%), but it isdifficult to isolate a sufficient quantity of betulinic acid to performan extensive bioassay. It has been found that a quantity of betulinicacid could be provided from betulin through a simple synthetic approach.A number of multi-step synthetic conversions of betulin to betulinicacid have been reported, but these synthetic sequences suffer from a lowoverall yield. A concise two-step conversion of betulin to betulinicacid, in good yield, has been reported in Synthetic Communications,27(9), pp. 1607-1612 (1997).

As shown in Table 1, in vitro growth of MEL-2 cells was inhibited bybetulinic acid, i.e., an ED₅₀ value of about 2 μg/ml. However, none ofthe other cancer cell lines tested was affected by betulinic acid (i.e.,ED₅₀ values of greater than 20 μg/ml). Such clearly defined cell-typespecificity demonstrated by betulinic acid is both new and unexpected.

For example, as illustrated in Table 1, other known antitumor agents,such as paclitaxel, camptothecin, ellipticine, homoharringtonine,mithramycin A, podopyllotoxin, vinblastine and vincristine, demonstratedrelatively intense, nonselective cytotoxic activity with no discerniblecell-type selectivity. Moreover, the cytotoxic response mediated bybetulinic acid is not exclusively limited to the MEL-2 melanoma cellline. Dose-response studies performed with additional human melanomacell lines, designated MEL-1, MEL-3 and MEL-4, demonstrated ED₅₀ valuesof 1.1, 3.3 and 4.8 μg/ml, respectively.

In the following Table 1, the extracted betulinic acid and the otherpure compounds were tested for cycotoxity against the following culturedhuman cell lines: A431 (squamous cells), BC-1 (breast), COL-2 (colon),HT-1080 (sarcoma), KB (human oral epidermoid carcinoma), LNCaP(prostate), LU-1 (lung), MEL-2 (melanoma), U373 (glioma) and ZR-75-1(breast).

TABLE 1 Cytotoxic Activity Profile of the Crude Ethyl Acetate ExtractObtained from Ziziphus mauritiana, Betulinic acid, Other AntineoplasticAgents ED₅₀ (μg/ml) Compound A431 BC-1 COL-2 HT-1080 KB LNCaP LU-1 MEL-2U373 ZR 75-1 Ziziphus mauritiana >20 >20 >20 9.5 >20 >20 5.2 3.7 >2015.8 crude extract Betulinic acid >20 >20 >20 >20 >20 >20 >202.0 >20 >20 Taxol 0.00 0.02 0.02 0.00 0.02 0.02 0.00 0.06 0.008 0.02Camptothecin 0.00 0.07 0.005 0.01 0.00 0.006 0.00 0.02 0.000 0.001Ellipticine 0.5 0.2 0.3 1.8 0.04 0.8 0.02 0.9 1.6 0.9 Homoharringtonine0.02 0.03 0.1 0.01 0.00 0.03 0.03 0.04 0.2 0.06 Mithramycin A 0.09 0.30.06 1.5 0.09 0.05 0.2 1.2 0.04 0.2 Podophyllotoxin 0.03 0.03 0.005 0.000.08 0.04 0.00 0.003 0.004 0.4 Vinblastine 0.05 0.06 0.01 0.02 0.04 0.10.02 0.01 1.1 0.3 Vincristine 0.01 0.01 0.02 0.02 0.00 0.1 0.05 0.020.06 0.4

Betulinic acid (1) has the structural formula:

Betulinic acid is fairly widespread in the plant kingdom, and, as acompound frequently encountered, some previous biological activitieshave been reported.

Betulinic acid was obtained by extracting a sample of air-dried, milledstem bark (450 g) of Z. mauritiana with 80% aqueous methanol. Theaqueous methanol extract then was partitioned successively with hexaneand ethyl acetate to provide hexane, ethyl acetate and aqueous extracts.Among these extracts, the ethyl acetate (13.5 g) extract showedcytotoxic activity against a cultured melanoma cell line (MEL-2) with anED₅₀ of 3.7 μg/ml. The ethyl acetate extract was chromatographed on asilica gel column using hexane-ethyl acetate (4:1 to 1:4) as eluent togive 10 fractions. Fractions 3 and 4 were combined and subjected tofurther fractionation to afford an active fraction (fraction 16) showinga major single spot by thin-layer chromatography [R_(f) 0.67: CHCl₃:MeOH(chloroform:methanol) (10:1)], which yielded 72 mg of colorless needlesafter repeated crystallization from methanol (overall yield from driedplant material: 0.0166 w/w).

As confirmed by the data summarized in Table 1, betulinic acid has beenreported as noncytotoxic with respect to cultured KB cells. Cytotoxicityof the crude extracts and purified compounds was determined in a numberof cultured human cancer cell lines. Table 1 sets forth the varioustypes of cancer cells evaluated. The cells were cultured in appropriatemedia and under standard conditions. To maintain logarithmic growth, themedia were changed 24 hours prior to cytotoxic assays. On the day of theassay, the cells were harvested by trypsinization, counted, diluted inmedia, and added to 96-well plates containing test compounds dissolvedin DMSO; the final DMSO concentration was 0.05%.

The plates were incubated for three days. Following the incubationperiod, the cells were fixed and stained with sulforhodamine B (SRB)dye. The bound dye was liberated with Tris base, and the OD₅₁₅ wasmeasured on an ELISA reader. The growth of the betulinic acid-treatedcells was determined by the OD₅₁₅ values, and the growth was compared tothe OD₅₁₅ values of DMSO-treated control cells. Dose response studieswere performed to generate ED₅₀ values.

The isolated active compound, betulinic acid (ED₅₀ of 2.0 μg/ml forMEL-2), has a molecular formula of C₃₀H₄₈O₃, as determined byhigh-resolution mass spectral analysis, a melting point range of292-293° C. (decomposition). The literature melting point range forbetulinic acid is 290-293° C. A mixed melting point range with a knownsample of betulinic acid was not depressed. The optical rotation of thecompound was measured as +7.3° (c=1.2; pyridine) (lit. +7.5°). Theidentity of the isolated compound as betulinic acid was confirmed bycomparing the above physical properties, as well as ¹H-nmr, ¹³C-nmr andmass spectral data of the isolated compound, with physical data andspectra of a known sample of betulinic acid as reported in theliterature.

To test the in vivo ability of betulinic acid to serve as anantineoplastic agent against malignant melanoma, a series of studies wasperformed with athymic (nude) mice injected subcutaneously with humanmelanoma cells (MEL-2). The initial study investigated the activity ofbetulinic acid against unestablished tumors. Treatment with betulinicacid began on day 1, i.e., 24 hours, following tumor cell injection. Atdoses of 50, 250, and 500 mg/kg (milligram per kilogram) body weight,betulinic acid demonstrated effective inhibition of tumor growth with pvalues of 0.001 for each dose versus a control (FIG. 1). These resultsindicate that betulinic acid can be used to prevent melanoma by topicalapplication of melanoma. Such a discovery is important for individualswho are predisposed to melanoma due to hereditary or environmentalfactors.

In particular, the data plotted in FIG. 1 was derived from experimentswherein four week old athymic mice were injected subcutaneously in theright flank with 3.0×10⁸ UISO MEL-2 cells. UISO MEL-2 is a cell linederived from metastatic melanoma from human pleural fluid. Drugtreatment was initiated on the day following tumor cell injection andcontinued every fourth day for a total of six doses. Four controlanimals received 0.5 ml intraperitoneal (IP) of PVP control solution,while treated animals (4 per group) received 50, 250 or 500 mg/kg/doseIP betulinic acid/PVP in deionized H₂O. Betulinic acid wascoprecipitated with PVP to increase solubility and bio-availability. Themice were weighed, and the tumors measured with a micrometer every otherday throughout the study. All animals were sacrificed and autopsied onday 33, when the mean tumor volume in the control animals wasapproximately one cm³.

There was greater inhibition of tumor growth at the highest dose ofbetulinic acid versus the lowest dose (p=0.04). Toxicity was ndtassociated with the betulinic acid treatment because toxicity isindicated by loss of body weight or other forms of acute toxicity. Noweight loss was observed.

Next, in vivo testing of betulinic acid was performed on establishedmelanomas. In this study, treatment was withheld until day 13, by whichtime a palpable tumor mass was present in all mice. As illustrated inFIG. 2, under these conditions betulinic acid successfully abrogatedtumor growth (p=0.0001). Furthermore, tumor growth did not parallel thatof the control (untreated) group even 14 days after the termination oftreatment.

In particular, with respect to FIG. 2, four-week-old athymic mice wereinjected with 5×10⁸ MEL-2 cells subcutaneously in the right flank. Fourtreatment groups of five mice each were studied. In one group, the micereceived 250 mg/kg/dose of IP betulinic acid/PVP every third day for sixtotal doses initiated the day following tumor cell injection. Thecontrol group received 0.5 ml IP saline. A DTIC treatment group received4 mg/kg/dose IP DTIC every third day from day 13 to day 28 of the study.The betulinic acid treatment group received 250 mg/kg/dose IP betulinicacid/PVP every third day from day 13 to day 27. The control andDTIC-treated mice were sacrificed and autopsied on day 36 due to theirlarge tumor burden. The remaining mice were sacrificed and autopsied onday 41.

As illustrated in FIG. 2, the efficacy of betulinic acid also wascompared to DTIC, which is clinically available for the treatment ofmetastatic melanoma. The dose of DTIC, which is limited by toxicity, wasselected to be equivalent to that administered to human patients. Tumorgrowth in the betulinic acid-treated group was significantly less thanthat observed in the DTIC-treated animals (p=0.0001). Compared tocontrols, DTIC produced a significant, but less pronounced, reduction intumor growth, with a p value of 0.01. A fourth group in this study wastreated with a schedule similar to that in the initial study. Underthese conditions, betulinic acid, as demonstrated before, significantlyinhibited tumor development (p=0.0001) and caused a prolonged reductionin tumor growth of up to three weeks following treatment termination.

FIGS. 4 and 5 illustrate that betulinic acid also showed activityagainst MEL-1 cells. In particular, with respect to FIGS. 4 and 5, fourweek old athymic mice were injected subcutaneously in the right flankwith 5.0×10⁸ UISO MEL-1 cells. Drug treatment was initiated on the dayfollowing tumor cell injection and continued every fourth day for atotal of six doses. Four control animals received 0.5 ml intraperitoneal(IP) saline, while treated animals (4 per group) received 5, 50 or 250mg/kg/dose IP betulinic acid/PVP in dd H₂O. The mice were weighed, andtumors were measured with a micrometer every third day throughout thestudy. Treated animals were sacrificed and autopsied on day 41, when themean tumor volume in the control mice was approximately 0.5 cm³. Thecontrol mice then received six doses of 50 mg/kg every fourth daybeginning day 41 and were sacrificed and autopsied on day 71.

The results illustrated in FIGS. 4 and 5 with respect to MEL-1 cellswere similar to the results illustrated in FIGS. 1 and 2. Betulinic acidtherefore is active both against MEL-1 and MEL-2 cells.

The mechanism by which antitumor agents mediated their activity is ofgreat theoretical and clinical importance. Therefore, the mode of actionby which betulinic acid mediates the melanoma-specific effect wasinvestigated. Visual inspection of melanoma cells treated with betulinicacid revealed numerous surface blebs. This observation, as opposed tocellular membrane collapse, suggested the induction of apoptosis. One ofthe most common molecular and cellular anatomical markers of apoptosisis the formation of “DNA ladders,” which correspond to the products ofrandom endonucleolytic digestion of internucleosomal DNA. Althoughrecent studies have shown that a lack of DNA laddering does notnecessarily indicate a failure to undergo apoptosis, double-strand DNAscission that yields a fragment of about 50 kilobase pairs (Kbp) hasbeen shown to consistently correlate with induction of apoptosis byvarious treatments in a variety of cell lines. Thus, generation of the50 Kbp fragment is a reliable and general indicator of apoptosis.Generation of the fragment occurs upstream of the process leading to DNAladders and represents a key early step in the commitment to apoptosis.

Therefore, an important feature of the present invention is a method ofanalyzing and quantifying the formation of the 50 Kbp fragment as abiomarker for induction of apoptosis in human cancer cell lines. Thismethod comprises treatment of cells in culture, followed by analysis ofthe total cellular DNA content using agarose field-inversion gelelectrophoresis. Under these conditions, the 50 Kbp fragment is resolvedas a diffuse band. The fraction of the total cellular DNA represented bythe 50 Kbp fragment is determined by densitometry on the contour of thisband.

To investigate the ability of betulinic acid to induce apoptosis, theabove-described method was adapted for use with the MEL-2 cell line. Asshown in FIG. 3A, time-dependent formation of a 50 Kbp DNA fragment wasinduced by betulinic acid with MEL-2 cells. Induction was at a maximumafter a 56 hour treatment period. After this time period, a decline inthe relative amount of the 50 Kbp fragment was observed, probably due tointernal degradation. Also observed in the agarose gel were DNAfragments of about 146 and about 194 Kbp, which are theorized to beprecursors in the process leading to the formation of the 50 Kbpfragment. Additionally, the induction of apoptosis (50 Kbp fragment)mediated by betulinic acid was dose-dependent (FIG. 3B), and the ED₅₀value (about 1.5 μg/ml) observed in the apoptotic response closelyapproximated the ED₅₀ value previously determined for the cytotoxicresponse (Table 1).

With further respect to FIG. 3A, cultured MEL-2 cells (10⁶ cellsinoculated per 25 cm² flask) were treated with 2 g/ml betulinic acid(200 μg/ml DMSO, diluted 1:100 in media) for 24, 32, 48, 56 and 72hours. After the treatment, the cells were harvested, collected bycentrifugation, then snap frozen in liquid nitrogen for subsequentanalysis. Samples were analyzed on a 1% agarose gel in a Hoefer HE100SuperSub apparatus cooled to 10° C. by a circulating water bath. Theelectrode buffer was 0.5×TBE buffer containing 0.25 μg/ml ethidiumbromide and was circulated during electrophoresis. Each gel included 20μL Sigma Pulse Marker 0.1-200 Kbp DNA size markers. Prior to sampleloading, 50 μL 2% SDS was added to each sample well. Each sample tubewas rapidly thawed, then the pelleted cells were immediately transferredin a volume about 50 μL to the well containing SDS. Each well then wasoverlaid with molten LMP agarose, which was allowed to gel prior toplacing the gel tray in the SuperSub apparatus.

Electrophoresis was performed at 172 volts for a total of 18 hours usingtwo sequential field inversion programs with pulse ramping. TheDNA/ethidium bromide fluorescence was excited on a UV transilluminatorand photographed using Polaroid type 55 P/N film. The negative wasanalyzed using a PDI scanning densitometer and Quantity One software.The intensity of the 50 Kbp fragment was determined by measuring thecontour optical density (OD×mm²) as a percent of the total opticaldensity in the sample lane, including the sample well. The decrease inthe 50 Kbp band definition caused by internal degradation, and does notrepresent a reversal of the process.

With further respect to FIG. 3B, cultured MEL-2 cells were treated for56 hours with the following concentrations of betulinic acid: 0, 0.1,1.0, 2.0, 4.0 and 8.0 μg/ml. The cells were harvested and apoptosismeasured as described for FIG. 3A. The experiment was repeated and asimilar dose-response curve was observed (data not shown).

These data suggest a causal relationship, and it is theorized thatbetulinic acid-mediated apoptosis is responsible for the antitumoreffect observed with athymic mice. Time-course experiments with humanlymphocytes treated in the same manner with betulinic acid atconcentrations of 2 and 20 μg/ml did not demonstrate formation of the 50Kbp fragment (data not shown) indicating the specificity and possiblesafety of the test compound.

Taking into account a unique in vitro cytotoxicity profile, asignificant in vivo activity, and mode of action, betulinic acid is anexceptionally attractive compound for treating human melanoma. Betulinicacid also is relatively innocuous toxicity wise, as evidenced byrepeatedly administering 500 mg/kg doses of betulinic acid withoutcausing acute signs of toxicity or a decrease in body weight. Betulinicacid was previously found to be inactive in a Hippocratic screen at 200and 400 mg/kg doses.

Betulinic acid also does not suffer from the drawback of scarcity.Betulinic acid is a common triterpene available from many speciesthroughout the plant kingdom. More importantly, a betulinic acid analog,betulin, is the major constituent of white-barked birch species (up to22% yield), and betulin can be converted to betulinic acid.

In addition to betulinic acid, betulinic acid derivatives can be used ina topically applied composition to selectively treat, or prevent orinhibit, a melanoma. Betulinic acid derivatives include, but are notlimited to esters of betulinic acid, such as betulinic acid esterifiedwith an alcohol having one to sixteen, and preferably one to six, carbonatoms, or amides of betulinic acid, such as betulinic acid reacted withammonia or a primary or secondary amine having alkyl groups containingone to ten, and preferably one to six, carbon atoms.

Another betulinic acid derivative is a salt of betulinic acid.Exemplary, but nonlimiting, betulinic acid salts include an alkali metalsalt, like a sodium or potassium salt; an alkaline earth metal salt,like a calcium or magnesium salt; an ammonium or alkylammonium salt,wherein the alkylammonium cation has one to three alkyl groups and eachalkyl group independently has one to four carbon atoms; or transitionmetal salt.

Other betulinic acid derivatives also can be used in the composition andmethod of the present invention. One other derivative is the aldehydecorresponding to betulinic acid or betulin. Another derivative isacetylated betulinic acid, wherein an acetyl group is positioned at thehydroxyl group of betulinic acid.

In particular, betulinic acid derivatives have been synthesized andevaluated biologically to illustrate that betulinic acid derivativespossess selective antitumor activity against human melanoma cells linesin vitro. It has been demonstrated that modifying the parent structureof betulinic acid provides numerous betulinic acid derivatives that canbe deused to prevent or inhibit malignant tumor growth, especially withrespect to human melanoma. The antitumor activity of betulinic acidderivatives is important because betulinic acid, although exhibiting ahighly selective activity against melanomas, also possesses a low watersolubility. The low water solubility of betulinic acid, however, can beovercome by providing an appropriate derivative of betulinic acid.Modifying the parent structure betulinic acid structure also can furtherimprove antitumor activity against human melanoma.

An examination of the structure of betulinic acid, i.e., compound (1),reveals that betulinic acid contains three positions, i.e., the C-3,C-20, and C-28 positions, where functional groups can be introduced. Inaddition, the introduced functional groups, if desired, then can bemodified. Through a series of reactions at these three positions, alarge number of betulinic acid derivatives were prepared and evaluatedfor bioefficacy against a series of human tumor cell lines, especiallyagainst human melanoma cell lines.

With respect to modifications at the C-3 position of betulinic acid, thehydroxyl group at the C-3 position can be converted to a carbonyl groupby an oxidation reaction. The resulting compound is betulonic acid,i.e., compound (2). The ketone functionality of betulonic acid can beconverted to oxime (3) by standard synthetic procedures. Furthermore, alarge number of derivatives (4) can be prepared through substitutionreactions performed on the hydroxyl group of oxime (3), withelectrophiles, as set forth in equation (a):

wherein R_(a)=H or C₁-C₁₆ alkyl, or R_(a)=COC₆H₄X, wherein X=H, F, Cl,Br, I, NO₂, CH₃, or OCH₃, or R_(a)=COCH₂Y, wherein Y=H, F, Cl, Br, or I,or R_(a)=CH₂CHCH₂ or CH₂CCR₁, wherein R₁ is H or C₁-C₆ alkyl. When R_(a)is C₁-C₁₆ alkyl, preferred alkyl groups are C₁-C₆ alkyl groups.

The ketone functionality of betulonic acid can undergo a reductiveamination reaction with various aliphatic and aromatic amines in thepresence of sodium cyanoborohydride (NaBH₃CN) to provide thecorresponding substituted amines (5) at the C-3 position, as set forthin equation (b).

wherein R_(b)=H or C₁-C₁₀ alkyl, or R_(b)=C₆H₄X. A primary aminederivative, i.e., R_(b)=H, at the C-3 position can be reacted with aseries of acyl chlorides or anhydrides, or alkyl halides, to provideamides and secondary amines (6), respectively, as set forth in equation(c).

wherein R_(c)=COC₆H₄X, or R_(c)=COCH₂Y, or R_(c)=CH₂CHCH₂ or CH₂CCR₁.

The ketone functionality of betulonic acid can react with a series oflithium acetylides (i.e., LiC≡CR₁) to provide alkynyl alcoholderivatives (7) at the C-3 position. Based on the chemical reactivity 5and the stereoselectivity of the betulonic acid structure, α-alkynylsubstituted β-hydroxyl alkynyl betulinic acid are the major products ofthe reaction, as set forth in equation (d).

wherein R_(d)=CCR₁, wherein R₁ is H or C₁-C₆ alkyl.

A number of esters also can be prepared by reacting the hydroxyl groupof betulinic acid with a variety of acyl chlorides or anhydrides (8), asset forth in equation (e).

wherein R_(e)=R₁CO or XC₆H₄CO.

With respect to modification at the C-28 position, the carboxyl group ofbetulinic acid can be converted to a number of esters (9) and amides(10) by reaction with an alcohol or an amine, respectively, as set forthin equations (f) and (g). Depending on the types of functional groupspresent on the alcohols or amines, additional structural modificationare possible. The carboxyl group also can be converted to a salt, inparticular an alkali metal salt, an alkaline earth salt, an ammoniumsalt, an alkylammonium salt, a hydroxyalkyl ammonium salt, or atransition metal salt.

wherein R_(f)=C₁-C₁₀ alkyl, phenyl, substituted phenyl (C₆H₄X), orCH₂CCR₁.

The activated C-28 hydroxyl group of betulin can undergo substitutionreactions, like SN-2 type reactions, with nucleophiles to provide anamino (11) or an ether derivative (12), as set forth in equations (h)and (i).

wherein R_(g)=H or C₁-C₁₆ alkyl, or R_(g)=C₆H₄X, and whereinR_(h)=C₁-C₁₆ alkyl or C₆H₄X.

The hydroxyl group at the C-28 position can be oxidized to yield analdehyde, which in turn can react with hydroxylamine to provide ahydroxyloxime compound. The hydroxyloxime can react with a variety ofelectrophiles to provide the oxime derivatives (13), as set forth inequation (j).

wherein R_(i)=H or C₁-C₁₆ alkyl, or R_(i)=COC₆H₄X, or R_(i)=COCH₂Y, orR_(i)=CH₂CHCH₂ or CH₂CCR₁.

The aldehyde at the C-28 position also can react with a series oflithium acetylide compounds to yield a variety of alkynyl betulinderivative (14), as set forth in equation (k).

wherein R_(j)=CCR₁, wherein R₁=H or C₁-C₆ alkyl.

With respect to modifications at the C-20 position, the isoprenyl groupat the C-20 position can be ozonized to yield a ketone (15) at C-20position, as set forth in equation (1). A variety of reactions performedon the ketone functionality can provide a series of differentderivatives. For example, the ketone functionality of compound (15) canbe easily converted to a variety of oximes. Furthermore, a number ofadditional oxime derivatives (16) can be prepared through substitutionreactions at the hydroxyl group of the hydroxyloxime with electrophiles,as set forth in equation (m).

wherein R_(k)=H or C₁-C₁₆ alkyl, or R_(k)=COC₆H₄X or R_(k)=COCH₂Y, orR_(k)=CH₂CHCH₂ or CH₂CCR₁.

The ketone functionality also can undergo a reductive amination reactionwith a series of aliphatic and aromatic amines in the presence ofNaBH₃CN to provide a corresponding substituted amine (17) at the C-20position, as set forth in equation (n).

wherein R_(l)=C₁-C₁₆ alkyl, or R_(l)=C₆H₄X, or R_(l)=COC₆H₄X, orR_(l)=COCH₂Y, or R_(l)=CH₂CHCH₂ or CH₂CCR₁.

The ketone can be reacted with a series of lithium acetylides to providealkynyl alcohol derivatives (18) at the C-20 position, as set forth inequation (o).

wherein, R_(m)=CCR₁.

The ketone further can be reduced to a secondary alcohol (19) to reactwith an acyl chloride to provide a series of esters (20) at the C-20position, as set forth in equation (p).

wherein R_(n)=H, C₁-C₁₆ alkyl, CH₂CCR₁, or R_(n)=CH₃CO or XC₆H₄CO.

In addition, a number of different derivatives can be prepared through acombinatorial chemical approach. For example, as set forth below, in thepreparation of oximes at the C-20 position, a number of electrophiles,e.g., a variety of alkyl halides, can be added together in one reactionvessel containing the hydroxyloxime to provide a mixture of betulinicacid derivatives. Each reaction product in the mixture can be isolatedby using semi-preparative HPLC processes using appropriate separationconditions, then submitted for bioassay.

wherein P is a protecting group for the secondary alcohol functionality.

A low temperature reaction of betulonic acid with a mixture of lithiumacetylides in a single reaction vessel, as set forth below, yielded amixture of alkynyl alcohols at the C-3 position. Each component in themixture can be isolated by using semi-preparative HPLC processes usingappropriate separation conditions, then submitted for bioassay.

In order to demonstrate that betulinic acid derivatives have a potentbioefficacy, various derivatives were subjected to a series ofbiological evaluation tests. The biological evaluation of thederivatives focused on the activity against human melanoma cell lines.In particular, the following betulinic acid derivatives were preparedand tested for cytotoxicity profile against human melanoma cell linesand against a number of selected nonmelanoma cell lines. The results aresummarized in Table 2. The data shows that some hydrogenatedderivatives, i.e., compounds 5 and 11, are less active thannonhydrogenated derivatives 13 and 10, respectively. However, otherhydrogenated derivatives, i.e., compounds 7 and 6, showed a comparablebiological activity to nonhydrogenated derivatives 2 and 8,respectively. Therefore, it is possible to optimize the modification atthe C-20 position to yield more potent betulinic acid derivatives. Table3 contains a summary of data showing the effect of hydrogenation at theC-20 position.

TABLE 2 Cytotoxicty Data of Betulinic Acid Derivatives

ED₅₀ [μg/mL] (Std. Dev.) Compound R₁ R₂ R₃ MEL-2 MEL-6 MEL-8 MALE-3M LOXKB  1 O═ CHO CH₂═C(CH₃)₂ 7.4 (2.4) >20 3.2 (1.2) >20 18.5 12.9  2 HO—N═COOH CH₂═C(CH₃)₂ 2.4 (0.3) 14.8 (2.0) 1.9 (1.0) 15.8 9.1 >20  3 CH₃O—N═CHNOCH₃ CH₂═C(CH₃)₂ >20 >20 >20 >20 >20 20  4 HO—N═ CHNOH CH₂═C(CH₃)₂2.2 (0.7) 11.9 (2.7) 1.4 (0.6) 17.5 4.1 3.3  5 CH₃O—N═ COOHC(CH₃)₃ >20 >20 >20 >20  6 O═ COOH C(CH₃)₃ 0.7 (0.6) 10.8 (2.6) 0.9(0.4) 20 (Dihydrobetulonic acid)  7 HO—N═ COOH C(CH₃)₃ 2.2 (0.3) 13.1(1.1) 1.6 (1.1) 13.9  8 O═ COOH CH₂═C(CH₃)₂ 0.9 (0.8) 15.3 (3.4) 0.4(0.1) 20 6.9 2.5 (Betulonic acid)  9 H₂N— COOH CH₂═C(CH₃)₂ 1.3 (0.4) 5.2(2.6) 1.3 (0.5) 3.1 10 HO— COOH CH₂═C(CH₃)₂ 1.2 (0.1) 13.2 (1.5) 1.0(0.3) 17.6 (0.5) >20 >20 (Betulinic acid) 11 HO— COOH C(CH₃)₃5.8 >20 >20 (Dihydrobetulinic acid) 12 HO— CH₂OH CH₂═C(CH₃)₂ >20 >20 >20(Betulin) 13 CH₃O—N═ COOH CH₂═C(CH₃)₂ 8.3 >20 4.3 14 HO— COOCH₃CH₂═C(CH₃)₂ 8.3 12.5 11.8 (Methyl betulinate) 15 HO— CH₃ CH₂═C(CH₃)₂17.6 15.6 >20 (Lupeol) 16 C₆H₄COO— CH₃ CH₂═C(CH₃)₂ >20 >20 >20 (Lupeolbenzoate) MEL-2, MEL-6, MEL-8, MALE-3M, and LOX are melanoma cell lines,and KB is human oral epidermoid carcinoma.

TABLE 3 Cytotoxicity Data of Betulinic Acid Derivatives (Effect ofHydrogenation at C-20)

ED₅₀ [μg/mL] (Std. Dev.) Compound R₁ R₂ R₃ MEL-2 MEL-6 MEL-8 MALE-3M LOXKB 13 CH₃O—N═ COOH CH₂═C(CH₃)₂ 8.3 >20 4.3  5 CH₃O—N═ COOHC(CH₃)₃ >20 >20 >20 >20 10 HO— COOH CH₂═C(CH₃)₂ 1.2 (0.1) 13.2 (1.5) 1.0(0.3) 17.6 (0.5) >20 >20 (Betulinic acid) 11 HO— COOH C(CH₃)₃5.8 >20 >20 (Dihydrobetulinic acid)  2 HO—N═ COOH CH₂═C(CH₃)₂ 2.4 (0.3)14.8 (2.0) 1.9 (1.0) 15.8 9.1 >20  7 HO—N═ COOH C(CH₃)₃ 2.2 (0.3) 13.1(1.1) 1.6 (1.1) 13.9  8 O═ COOH CH₂═C(CH₃)₂ 0.9 (0.8) 15.3 (3.4) 0.4(0.1) 20 6.9 2.5 (Betulonic acid)  6 O═ COOH C(CH₃)₃ 0.7 (0.6) 10.8(2.6) 0.9 (0.4) 20 (Dihydrobetulonic acid)

The modification of betulinic acid at the C-3 position showed that allcompounds, except methoxy oxime 13, expressed a comparable biologicalactivity toward melanoma cell lines (Table 4). Amino compound 9exhibited an improved cytotoxicity compared to betulinic acid 10.Compounds 2, 8, and 13 showed a decrease in selective cytotoxicitycompared to betulinic acid.

TABLE 4 Cytotoxicity Data of Betulinic Acid Derivatives (Modification atC-3 Position)

ED₅₀ [μg/mL] (Std. Dev.) Compound R₁ R₂ R₃ MEL-2 MEL-6 MEL-8 MALE-3M LOXKB 10 HO— COOH CH₂═C(CH₃)₂ 1.2 (0.1) 13.2 (1.5) 1.0 (0.3) 17.6(0.5) >20 >20 (Betulinic acid)  8 O═ COOH CH₂═C(CH₃)₂ 0.9 (0.8) 15.3(3.4) 0.4 (0.1) 20 6.9 2.5 (Betulonic acid)  2 HO—N═ COOH CH₂═C(CH₃)₂2.4 (0.3) 14.8 (2.0) 1.9 (1.0) 15.8 9.1 >20 13 CH₃O—N═ COOH CH₂═C(CH₃)₂8.3 >20 4.3  9 H₂N— COOH CH₂═C(CH₃)₂ 1.3 (0.4)  5.2 (2.6) 1.3 (0.5)  3.1

With respect to modifications at the C-28 position, the free carboxylicacid group at C-28 position is important with respect to expression ofbiological activity (Table 5). However, it is unknown whether the sizeor the strength of hydrogen bonding or the nucleophilicity of the C-28substituents is responsible for the biological effect.

TABLE 5 Cytotoxicity Data of Betulinic Acid Derivatives (Modification atC-28 Position)

ED₅₀ [μg/mL] (Std. Dev.) Compound R₁ R₂ R₃ MEL-2 MEL-6 MEL-8 MALE-3M LOXKB 12 HO— CH₂OH CH₂═C(CH₃)₂ >20 >20 >20 (Betulin) 10 HO— COOHCH₂═C(CH₃)₂ 1.2 (0.1) 13.2 (1.5) 1.0 (0.3) 17.6 (0.5) >20 >20 (Betulinicacid) 14 HO— COOCH₃ CH₂═C(CH₃)₂ 8.3 12.5 11.8 (Methyl betulinate) 15 HO—CH₃ CH₂═C(CH₃)₂ 17.6 15.6 >20 (Lupeol)

The biological activity changes attributed to oximes is illustrated inTable 6. The hydroxyloxime 4 improved the cytotoxicity profile, althoughselectivity was lost. It appears that the size of the substituent andits ability to hydrogen bond may influence the expression of thebiological activity.

TABLE 6 Cytotoxicity Data of Betulinic Acid Derivatives (Effect byOximes)

ED₅₀ [μg/mL] (Std. Dev.) Compound R₁ R₂ R₃ MEL-2 MEL-6 MEL-8 MALE-3M LOXKB 12  HO— CH₂OH CH₂═C(CH₃)₂ >20 >20 >20 (Betulin) 1 O═ CHO CH₂═C(CH₃)₂7.4 (2.4) >20 3.2 (1.2) >20 18.5 12.9 4 HO—N═ CHNOH CH₂═C(CH₃)₂ 2.2(0.7) 11.9 (2.7) 1.4 (0.6) 17.5 4.1 3.3 3 CH₃O—N═ CHNOCH₃CH₂═C(CH₃)₂ >20 >20 >20 >20 >20 20

The above tests show that modifying the parent structure of betulinicacid can provide derivatives which can be used as potent antitumor drugsagainst melanoma. Betulinic acid derivatives having a comparable orbetter antitumor activity than betulinic acid against human melanomahave been prepared. In addition, even though betulinic acid has aremarkably selective antitumor activity, betulinic acid also has a poorsolubility in water. The low solubility of betulinic acid in water canbe overcome by introducing an appropriate substituent on the parentstructure, which in turn can further improve selective antitumoractivity. In addition, because the parent compound, betulinic acid, hasshown to possess anti-HIV activity, the derivatives also can bedeveloped as potential anti-HIV drug candidates.

What is claimed is:
 1. A method of inhibiting growth of a melanomacomprising topically applying a therapeutically effective amount of acomposition comprising an effective amount of betulinic acid modified atthe C-28 position and having the structure

wherein R_(f) is C₁-C₁₀ alkyl, phenyl, or C₆H₄X, and wherein X is H, F,Cl, Br, I, NO₂, CH₃, or OCH₃ to the melanoma.
 2. The method of claim 1wherein R_(f) is CH₃.
 3. A method of preventing melanoma comprisingtopically applying a therapeutically effective amount of a compositioncomprising an effective amount of betulinic acid modified at the C-28position and having the structure

wherein R_(f) is C₁-C₁₀ alkyl, phenyl, or C₆H₄X, and wherein X is H, F,Cl, Br, I, NO₂, CH₃, or OCH₃ to skin in need thereof.
 4. The method ofclaim 3 wherein R_(f) is CH₃.